FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis.

Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis.

Analyzing the function of a hox gene: an evolutionary approach.

We present an evolutionary approach to dissecting conserved developmental mechanisms. We reason that important mechanisms for making the bodyplan will act early, to generate the major features of the body and that they will be conserved in evolution across many metazoa, and thus, that they will be available in very different animals. This led to our specific approach of microarrays to screen for very early conserved developmental regulators in parallel in an insect, Drosophila and a vertebrate, Xenopus. We screened for the earliest conserved targets of the ectopically expressed hox gene Hoxc6/Antennapedia in both species and followed these targets up, using in situ hybridization, in the Xenopus system. The results indicate that relatively few of the early Hox target genes are conserved: these are mainly involved in the specification of the antero-posterior body axis and in gastrulation.

Axial patterning in snakes and caecilians: evidence for an alternative interpretation of the Hox code.

It is generally assumed that the characteristic deregionalized body plan of species with a snake-like morphology evolved through a corresponding homogenization of Hox gene expression domains along the primary axis. Here, we examine the expression of Hox genes in snake embryos and show that a collinear pattern of Hox expression is retained within the paraxial mesoderm of the trunk. Genes expressed at the anterior and most posterior, regionalized, parts of the skeleton correspond to the expected anatomical boundaries. Unexpectedly however, also the dorsal (thoracic), homogenous rib-bearing region of trunk, is regionalized by unconventional gradual anterior limits of Hox expression that are not obviously reflected in the skeletal anatomy. In the lateral plate mesoderm we also detect regionalized Hox expression yet the forelimb marker Tbx5 is not restricted to a rudimentary forelimb domain but is expressed throughout the entire flank region. Analysis of several Hox genes in a caecilian amphibian, which convergently evolved a deregionalized body plan, reveals a similar global collinear pattern of Hox expression. The differential expression of posterior, vertebra-modifying or even rib-suppressing Hox genes within the dorsal region is inconsistent with the homogeneity in vertebral identity. Our results suggest that the evolution of a deregionalized, snake-like body involved not only alterations in Hox gene cis-regulation but also a different downstream interpretation of the Hox code.

The role of the Spemann organizer in anterior-posterior patterning of the trunk.

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.

Kinetics of glucocorticoid exposure in developing zebrafish: A tracer study.

In the current study the dynamics of glucocorticoid uptake by zebrafish chorionated embryos from the surrounding medium were studied, using 2.5 µM cortisol or dexamethasone solutions complemented with their tritiated variant. We measured the uptake of radioactive cortisol by embryos during a 1 h submersion. Interestingly, the signal in chorionated embryos was 85% (exposure: 1-2 hpf) or 78% (exposure: 48-49 hpf) of the signal present in an equal volume medium. By comparing embryos measured without chorion, we found that 18-20% of the radioactivity present in chorionated embryos is actually bound to the chorion or located in the perivitelline space. Consequently, embryonic tissue contains radioactivity levels of 60% of a similar volume of medium after 1 h incubation. During early developmental stages (1-48 hpf) exposure of more than 24 h in cortisol was needed to achieve radioactivity levels similar to an equal volume of medium within the embryonic tissue and more than 48 h for dexamethasone. In glucocorticoid-free medium, radioactivity dropped rapidly below 10% for both glucocorticoids, suggesting that the major portion of the embryonic radioactivity was a result of simple diffusion. During later developmental stages (48-96 hpf) initial uptake dynamics were similar, but showed a decrease of tissue radioactivity to 20% of an equal volume of medium after hatching, probably due to development and activation of the hypothalamic pituitary interrenal axis. Uptake is dependent on the developmental stage of the embryo. Furthermore, the presence of the chorion during exposure should be taken into account even when small lipophilic molecules are being tested.

Vertical signalling involves transmission of Hox information from gastrula mesoderm to neurectoderm.

Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of Xenopus laevis. Our results show that copying of positional information involves non cell autonomous autoregulation of particular Hox genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the Hox pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved Hox genes.

Two Hoxc6 transcripts are differentially expressed and regulate primary neurogenesis in Xenopus laevis.

Hox genes are key players in defining positional information along the main body axis of vertebrate embryos. In Xenopus laevis, Hoxc6 was the first homeobox gene isolated. It encodes two isoforms. We analyzed in detail their spatial and temporal expression pattern during early development. One major expression domain of both isoforms is the spinal cord portion of the neural tube. Within the spinal cord and its populations of primary neurons, Hox genes have been found to play a crucial role for defining positional information. Here we report that a loss-of-function of either one of the Hoxc6 products does not affect neural induction, the expression of general neural markers is not modified. However, Hoxc6 does widely affect the formation of primary neurons within the developing neural tissue. Manipulations of Hoxc6 expression severly changes the expression of the neuronal markers N-tubulin and Islet-1. Formation of primary neurons and formation of cranial nerves are affected. Hence, Hoxc6 functions are not restricted to the expected role in anterior-posterior pattern formation, but they also regulate N-tubulin, thereby having an effect on the initial formation of primary neurons in Xenopus laevis embryos.

Knockdown of the complete Hox paralogous group 1 leads to dramatic hindbrain and neural crest defects.

The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.